Sucrose accumulating tomato technology

ABSTRACT

A new tomato cultivar is provided which is homozygous for a genetically fixable recessive genetic factor which confers the ability to bear fruit that accumulate sucrose. The increased sucrose content, in turn, leads to a higher total soluble solids content in the mature fruit. Higher total soluble solids in tomato fruit are of particular importance to the tomato processing industry. A method for producing the claimed cultivar is also provided. 
     The claimed genetic factor can be derived from any Lycopersicon species which accumulates sucrose in the mature fruit. Once genetically fixed in a tomato cultivar, the factor can be transmitted to other plants in a predictable manner.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the breeding of tomato plants. More specifically, the invention relates to the introduction of a genetic factor that confers on tomato plants the ability to bear mature fruit which accumulate sucrose, thereby increasing the total soluble solids content of the fruit.

2. Information Disclosure

In the prior art, increasing the soluble solids content of tomato fruit has been attempted by introducing the high soluble solids trait from wild Lycopersicon species. Rick, Hilgardia, 42:494-509 (1974), used L. chmielewskii in a breeding plan to produce a variety, known as LA 1563, which showed an increase in soluble solids content. Genetic analysis of this variety indicated that the high soluble solids trait was dominant and was controlled by a number of loci. Restriction fragment length polymorphisms have also been used to create linkage maps of the loci that are associated with the trait. Paterson et al., Nature, 335:721-726 (1988).

Investigations of carbohydrate metabolism in tomato reveal that fruit of tomato cultivars accumulate glucose and fructose, but not sucrose. Yelle et al., Plant Physiol, 87:737-740 (1988). The rate of carbohydrate import, a determinant of soluble solids content of the tomato fruit, has been reported to be inversely proportional to sucrose concentration in the fruit. Walker and Thornley, Ann. Bot., 41:977-985 (1977).

The fruit of three other Lycopersicon species (L. chmielewskii, L. hirsutum, and L. peruvianum) have been shown to have a high soluble solids content. Accessions of these species accumulate sucrose instead of glucose and fructose. Davies, Nature, 209:640-641 (1966). The prior art, however, provided no teaching as to whether this trait was under the control of one or many loci. Studies also show that these species have very low acid invertase level compared to L. esculentum. Manning and Maw, Phytochemistry, 14:1965-1969 (1975) and Yelle at al., supra. The fruit of the high solids variety produced by Rick, LA 1563, has been shown not to accumulate sucrose. Hewitt et al., J. Amer. Soc. Hort. Soc., 107, 896-900 (1982). This result suggests that the L. chmielewskii trait for sucrose accumulation was not genetically fixed in LA 1563.

SUMMARY OF THE INVENTION

The present invention provides a tomato plant which is homozygous for a single, recessive genetic factor which confers on the plant the ability to bear mature fruit which accumulate much more sucrose than hexose sugars. Generally, the sucrose content of the fruit will be between about 20 μmol per gram fresh weight and about 175 μmol per gram fresh weight, preferably from about 50 μmol to about 150 μmol, and most preferably about 100 μmol.

As a result of the increased sucrose the mature fruit of the claimed plant have an increased total soluble solids content. The soluble solids content is generally between about 6% by fresh weight and about 12% by fresh weight, preferably from about 7.5% to about 10%, and most preferably about 8%.

The fruit of the claimed plant have a lower level of invertase activity than standard tomato cultivars. Typically, the invertase activity is less than about 5 μmol of glucose produced per gram of fresh weight in one minute. Invertase activity is preferably less than about 1 μmol and most preferably about 35 nmol.

The genetic factor can be derived from any sucrose accumulating Lycopersicon species. It is preferably derived from L. chmielewskii.

The invention also provides a method for introducing into a tomato plant a single, recessive genetic factor which confers on the tomato plant the ability to bear a mature fruit having an total soluble solids content of about 8% by fresh weight. The method comprises the steps of crossing the tomato plant with a second plant in the genus Lycopersicon having the genetic factor, thereby producing an F1 generation; selfing the F1 plants, thereby producing an F₂ generation; selecting F₂ plants which produce mature fruit having a sucrose content of about 100 μmol per gram fresh weight; and back-crossing the selected F₂ plants with L. esculentum, thereby producing a BC₁ F₁ generation; selfing the BC₁ F₁ plants, thereby producing a BC₁ F₂ generation; selecting BC₁ F₂ plants with the desired phenotype and backcrossing with L. esculentum as the recurrent parent until an agromonically useful cultivar is produced. The second plant can be either L. chmielewskii or L. esculentum. If the second plant is L. esculentum, it is typically a tomato cultivar.

The present invention also provides a genetically fixed genetic factor which confers on a tomato plant the ability to bear a mature fruit which accumulate sucrose in place of hexose sugars. Generally, the sucrose content of the fruit will be between about 20 μmol per gram fresh weight and about 175 μmol per gram fresh weight, preferably about 100 μmol.

The mature fruit of a plant expressing the claimed factor exhibit an increased total soluble solids content. The soluble solids content is generally between about 6% by fresh weight and about 12% by fresh weight, preferably about 8%.

The fruit of the plant expressing the claimed factor have a lower level of invertase activity than standard tomato cultivars. Typically, the invertase activity is less than about 5 μmol of glucose produced per gram of fresh weight per minute.

The genetic factor can be derived from any sucrose accumulating Lycopersicon species. It is preferably derived from L. chmielewskii.

The present invention also provides tomato seed homozygous for the genetic factor. Plants derived from the seed have a sucrose content from about 20 μmol per gram fresh weight to about 175 μmol per gram fresh weight. The plants derived from the seed also exhibit increased total soluble solids and decreased invertase activity in the mature fruit.

DETAILED DESCRIPTION

Improved tomato plants (Lycopersicon esculentum) characterized by a genetically fixable and recessive genetic factor which confers the ability to bear fruit having an increased total soluble solids are provided. The genetic factor is derived from Lycopersicon species which accumulate sucrose in the mature fruit. The genetic factor acts substantially as a recessive gene or locus which can be transmitted between tomato cultivars by conventional breeding techniques.

Soluble solids are of major economic significance to the tomato processing industry. For example, tomato paste must contain at least 24% soluble solids. Standard tomato cultivars, however, generally have a total soluble solids content of between 5 and 7%. Hewitt and Garrey, in Tomato Biotechnology, pp. 45-54 (Nevins and Jones, eds. 1987). It is estimated that a savings of $7 million can be realized by the tomato processing industry for every 0.1% increase in soluble solids content. Kunimoto, Food Tech., pp. 58-60 (October, 1986).

While not wishing to be bound by theory, increased total soluble solids content in the fruit of tomato plants expressing the claimed genetic factor apparently results from increased accumulation of sucrose in the mature fruit. Sucrose accumulation is apparently the result of decreased activity of the enzyme, acid invertase, which catalyzes the hydrolysis of sucrose to glucose and fructose. Thus, sucrose, rather than glucose and fructose, accumulates in the fruit of plants expressing the claimed genetic factor. Sucrose accumulation presumably leads to higher total soluble solids for two reasons. First, sucrose is not as readily metabolized as glucose and fructose. Unless sucrose is cleaved, it is stored as a metabolic end product and is not consumed through respiration. Second, because sucrose is a disaccharide, twice as much sucrose as glucose and fructose can accumulate, while maintaining the same turgor pressure.

The method of the present invention is not limited to the particular factor which has been isolated. Any Lycopersicon species which accumulates sucrose in the mature fruit can be the source of the claimed genetic factor. The genetic factor isolated from L. chmielewskii is preferred because crosses between that species and tomato cultivars can be readily made. Other species that could be used as a source for the claimed genetic factor include L. hirsutum, L. hirsutum var. glabratum, and L. peruvianum.

The methods of the present invention can be used to modify and improve the soluble solid content of all tomato cultivars. Open pollinated cultivars that can be used include Hunt 100, UC204, E6203, UC82B, Advantage, and HM3075. Hybrid cultivars that can be used include, FM785, Alta, Lassen, Brigade, N1400, N1401 and N1200.

The method of the present invention can be used to produce tomato cultivars for commercial tomato production. Any standard method used for crossing tomato plants can be used. Generally, the methods involve emasculation of one parent, followed by application of pollen from the other parent to the stigma of the first parent. The crosses can be performed using either parent as the pollen parent. Embryo rescue can also be performed if the flowers abort after pollination.

A tomato plant of the present invention can be obtained by crossing a plant homozygous for the claimed genetic factor with any tomato cultivar lacking the factor. The plant containing the factor can be any Lycopersicon species, including a tomato cultivar in which the factor has been previously genetically fixed.

Because the genetic factor is recessive, the F₁ generation will not accumulate sucrose in the mature fruit. Only a tomato plant homozygous for the genetic factor will exhibit the sucrose-accumulating phenotype. This phenotype can be used to identify progeny that are homozygous for the claimed genetic factor.

After selfing the F₁ population, the F₂ generation will exhibit the phenotype in a ratio of approximately 1:4. Back-crossing F₂ sucrose-accumulating individuals with L. esculentum plants will produce the BC₁ F₁ population. Selfing the BC₁ F₁ population will give the BC₁ F₂ generation. As in the F₂ population, the sucrose-accumulating trait will segregate in a ratio of about 1:4 in this population. Repeated back-crosses will produce a sucrose-accumulating tomato cultivar with the characteristics of the recurrent, parent cultivar. The claimed genetic factor will thus become generally fixed in the resulting cultivar. The trait may then be transmitted by sexual crossing to other cultivars, if desired.

Of course, other breeding schemes can be used to introduce the genetic factor into the desired cultivar. The particular scheme used is not critical to the invention, so long as the genetic factor is stably incorporated into the genome of the cultivar. For instance, other marker genes such as TG102, which is associated with sterility of flowers in crosses between L. esculentum and L. chmielewskii, can be used. TG102 is apparently tightly linked to the claimed genetic factor (see experimental section, infra). A nucleic acid probe which hybridizes to the TG102 marker gene can be used to identify the desired plants in the F₁ generation. This approach would eliminate the need for selfing the progeny of each backcross.

In order to determine if a cultivar possesses the claimed genetic factor, a classic genetic test for allelism can be performed. The cultivar is crossed with a plant known to possess the claimed genetic factor and to exhibit the sucrose accumulating phenotype. By analyzing the resulting F₁ generation, the genotype of the unknown cultivar can be determined. If the unknown cultivar possesses the genetic factor, the sucrose-accumulating phenotype will be observed in the F₁ generation. Nonallelic genetic factors that also result in sucrose accumulation can be identified by analyzing the sugar composition of the unknown cultivar.

A tomato plant having the high soluble solids phenotype can be characterized by its ability to transmit the phenotype to progeny when crossed with a normal tomato cultivar that lacks the claimed genetic factor. In addition, plants with the high soluble solids phenotype will display certain well defined characteristics. First, the fruit will have total soluble solids content of about 8%. Total soluble solids are typically measured as °Brix. °Brix is a standard refractometric measure of soluble solids which primarily detects reducing sugars, but is also affected by other soluble constituents. 1° Brix is approximately 1% by weight.

Second, the fruit will accumulate much more sucrose than hexose sugars and have a sucrose content of about 100 μmol per gram fresh weight. The sugar content of the fruit is typically determined using the arsenomolybdate based assay for reducing sugars described in Nelson, J. Biol. Chem., 153:375-380 (1944) which is incorporated herein by reference. Sucrose content can be determined by the difference in values obtained from samples incubated in the presence and absence of invertase.

Third, the acid invertase levels in the fruit will be low, generally about 35 nmol of glucose produced per gram of fresh weight in one minutes. Enzyme activities are typically determined by the method of Robinson et al., Plant Physiol., 87:727-730 (1988), which is incorporated herein by reference. According to this method, endogenous hexose sugars are removed form crude extracts of tomato fruit. The samples are incubated for 30 minutes and the reducing sugars produced are measured using the assay of Nelson, supra.

DEFINITIONS

The phrase "total soluble solids" refers to the soluble portion of the dry matter of Lycopersicon fruit. The soluble solids are composed of carbohydrates, the major components of which are starch and sugars. In Lycopersicon fruit, the sugars are primarily the hexoses, fructose and glucose, and the disaccharide, sucrose.

The term "recessive" is used to refer to an allele whose phenotypic effect is masked in a heterozygote by another, dominant, allele. Thus, the heterozygote is indistinguishable phenotypically from individuals homozygous for the dominant allele.

The term "homozygous" refers to condition in which a diploid individual carries identical alleles at a given genetic locus.

The term "genetic factor" refers to a genetic locus flanked by at least one non-wild type region which is stably incorporated into the genome of a plant and which confers on the plant a characteristic phenotype.

The term "fresh weight" refers to the total weight of plant tissue, it includes both dry matter and water.

The term "mature fruit" refers to fully ripe tomato fruit with a total lycopene content (the primary pigment in ripe fruit) of about 40-70 μgrams per gram fresh weight. Maturity is typically reached about 40 to 50 days after anthesis.

The term "cultivar" refers to a commercially valuable, horticulturally derived, variety, as distinguished from a naturally occurring variety.

The term "genetically fixed" refers to a genetic factor which has been stably incorporated into the genome of a plant that normally does not contain the genetic factor. When genetically fixed, the genetic factor can be transmitted in a predictable manner to other plants by sexual crosses.

The term "sucrose-accumulating" refers to a phenotype characterized by the ability of the mature fruit to accumulate much more sucrose than hexose sugars.

The following experimental results are offered by way of example and not by way of limitation.

EXAMPLES I. Segregation of Sucrose-Accumulating Trait

L. esculentum cv. UC 82 (available from Department of Vegetable Crops, University of California, Davis) and L. chmielewskii (LA 1028, available from the Tomato Genetic Stock Center, University of California, Davis) were crossed using standard methods of pollination and emasculation. The resulting F₁ hybrids were selfed to yield a population of F₂ progeny that were analyzed for sugar composition.

For sugar determination, 3 g of ripe fruit tissue was homogenized in 80% ethanol and heated to 100° C. for 20 minutes and centrifuged to remove insoluble material. Replicate samples were analyzed for reducing sugars using the arsenomolybdate based assay of Nelson, supra. Sucrose concentration was determined by incubating the sugars of each sample in the presence of yeast invertase at 37° C. for 30 minutes. (Yeast invertase was obtained from Sigma Chemical Co., St. Louis, Mo.). Sucrose content was then inferred by comparing the sugar concentration of samples in the presence and absence of invertase. The raw data for sucrose concentration obtained by this method were expressed in equivalent glucose concentration (μmol/gr fr wt). Because sucrose is a disaccharide, the actual sucrose concentration in each fruit is one-half the value obtained.

In the F₂ population, 6 of 91 plants analyzed bore fruit that accumulated sucrose as the predominant sugar. A sucrose accumulating plant was backcrossed with L. esculentum cv. UC 204B (available from Department of Vegetable Crops, University of California, Davis). This cross produced a population of BC₁ F₁ progeny which were selfed to produce the BC₁ F₂ population that was again analyzed for sugar composition. UC 204B was used as the recurrent parent in all subsequent backcrosses.

Analysis of sugar composition of the BC₁ F₂ population gave the results as summarized in Table 1.

                  TABLE I                                                          ______________________________________                                         Sugar composition of BC.sub.1 F.sub.2 fruit and UC82.                                      Hexose     Sucrose  Total Sugar                                                μmol/gr μmol/gr                                                                              μmol/gr                                     Population  fr wt      fr wt    fr wt                                          ______________________________________                                         UC82 N = 46 165.9       6.4     178.7                                          BC.sub.1 F.sub.2 (Sucrose                                                      Accumulators)                                                                  4005-4      19.2       168.9    326.9                                          4005-5      38.2       97.4     232.9                                          4079-5      48.4       107.8    263.9                                          4077-13     55.7       86.1     227.8                                          4006-27     69.0       81.5     232.0                                          4005-1      83.8       84.4     252.6                                          4077-7      83.3       55.6     194.4                                          4077-6      83.8       51.4     186.5                                          4079-7      120.3      70.0     260.2                                          4006-18     117.1      37.2     191.4                                          X           71.9       82.5     236.9                                          ______________________________________                                          Total Sugar content expressed as equivalent glucose units.               

The ratio of sucrose to hexose-accumulating plants in the BC₁ F₂ population was 1:5.6 (10 plants out of 56), rather than the predicted 1:4 for a trait determined by a single, recessive gene. Restriction fragment length polymorphisms were utilized to map the chromosomal location of the genetic factor conferring sucrose accumulation and found to be localized adjacent to a DNA marker, TG102, on chromosome 3. This region is also associated with sterility of flowers in crosses between Lycopersicon esculentum and Lycopersicon chmielewskii. It was found that six BC₁ F₂ plants scored as homozygous for the L. chmielewskii allele of TG102. These plants were sterile and thus excluded from analysis although they were likely to be sucrose-accumulators. This consideration suggests that the true ratio of sucrose accumulating plants would have been 1:3.87, consistent with genetic control by a single recessive gene. Because the gene is tightly linked to TG102, the proportion of plants exhibiting the phenotype was artificially reduced.

II. Determination of Total Soluble Solids Content

Total soluble solids content was measured as °Brix. An extract for measuring soluble solids was prepared by mechanically homogenizing fruit and filtering the homogenate through cheesecloth. The refractive index of the filtrate was then determined using a Bausch & Lomb refractometer. The results are presented below in Table II.

                  TABLE II                                                         ______________________________________                                         Total Soluble Solids in BC.sub.1 F.sub.2 fruit and UC 82.                             Plant    °Brix                                                   ______________________________________                                                UC82     5                                                                     BC.sub.1 F2.sub.1                                                              (Sucrose                                                                       accumulators)                                                                  4005-4   10.8                                                                  4005-5   N.D.                                                                  4079-5   10.2                                                                  4077-13  8.6                                                                   4006-27  N.D.                                                                  4005-1   9.0                                                                   4077-7   8.2                                                                   4077-6   7.5                                                                   4079-7   7.9                                                                   4006-18  N.D.                                                                  Average  8.9                                                            ______________________________________                                    

III. Determination of Invertase and Sucrose Synthase Activities

Activities of two enzymes potentially involved in sucrose breakdown were assayed and it was determined that invertase was absent in sucrose accumulating plants but present at high levels in hexose-accumulating tomato fruit.

Invertase and sucrose synthase activities were determined according to the method of Robinson, et al. supra. Briefly, crude extracts from tomato fruit were centrifuged through a 1 ml column of Sephadex G-50 to remove endogenous sugars. The column was washed twice with to 50 mM Hepes-KOH (pH 8.3), followed by two washes with 50 mM Hepes-KOH (pH 8.3) containing 0.1% BSA.

Invertase activity was determined in an assay containing 0.1 ml 6% sucrose in 1M Na acetate (pH 4.5) and desalted extract in a final volume of 0.2 ml. The reaction was terminated by heat denaturation after 30 minutes at 37° C. The products were determined using the assay of Nelson, Supra.

The sucrose synthase assay contained 0.25 μmol UDP[¹⁴ C] glucose (160,000-240,000 cpm/μmol) 0.5 μmol fructose, 5 μmol Tris-HCl (pH 8.0), and desalted extract in a final volume of 0.05 ml. The reaction was terminated after 20 minutes at 30° C. Dowex-1 formate was used to bind unreacted UDP[¹⁴ C] glucose and [¹⁴ C] sucrose was measured by standard methods.

The results of these assays are presented below in Table III.

                  TABLE III                                                        ______________________________________                                         Invertase and sucrose synthase activities in                                   BC.sub.1 F.sub.2 fruit and UC82 fruit.                                                  Days after                                                                               Invertase activity                                                                          Sucrose Synthase                                        anthesis  (nmol/gr     activity (nmol/gr                              Population                                                                              (days)    fr wt min)   fr wt min)                                     ______________________________________                                         UC82     20        3216 ± 271                                                                               397 ± 52                                    N = 46   40        12839 ± 572                                                                              N.D.*                                          BC.sub.1 F.sub.2                                                                        20         42 ± 20  346 ± 57                                    N = 10   40         32 ± 29  N.D.*                                          ______________________________________                                          *Sucrose synthase levels not determined (N.D. at 40 days after anthesis        since levels are very low in both L. esculentum and L. chmielewskii at         this developmental stage.                                                 

What is claimed is:
 1. A method for introducing into a Lycopersicon esculentum plant a single, recessive genetic factor which confers on the tomato plant the ability to bear a mature fruit having a sucrose content of about 100 μmol per gram fresh weight, the method comprising:a. crossing the tomato plant with a second plant in the genus Lycopersicon, which is homozygous for the genetic factor, thereby producing an F₁ generation; b. selfing the F₁ plants, thereby producing an F₂ generation; c. selecting F₂ plants which produce mature fruit having a sucrose content of a bout 100 μmol per gram fresh weight; d. back-crossing the selected F₂ plants with L. esculentum, thereby producing a BC₁ F₁ generation; e. selfing the BC₁ F₁ plants, thereby producing a BC₁ F₂ generated; f. selecting BC₁ F₂ plants which produce mature fruit having a sucrose content of about 100 μmol per gram fresh weight, and which are homozygous for a TG102 allele associated with the second plant.
 2. A method according to claim 1 wherein the second plant is Lycopersicon chmielewskii.
 3. A method according to claim 1 wherein the second plant is Lycopersicon esculentum.
 4. A method according to claim 1 wherein the second plant is a tomato cultivar.
 5. A genetically fixed, recessive genetic factor which confers on a Lycopersicon esculentum plant the ability to bear a mature fruit having sucrose content from about 20 μmol per gram fresh weight to about 175 μmol per gram fresh weight, wherein the genetic factor is located adjacent a TG102 marker on chromosome
 3. 6. A genetic factor according to claim 5 wherein the mature fruit has a sucrose content of about 100 μmol per gram fresh weight.
 7. A genetic factor according to claim 5 wherein the mature fruit has a total soluble solids content from about 6% by fresh weight to about 12% by fresh weight.
 8. A genetic factor according to claim 5 wherein the fruit has a level of invertase activity which is less than about 5 μmol of sucrose per gram of fresh weight per minute.
 9. A genetic factor according to claim 5 which is derived from Lycopersicon chmielewskii.
 10. A Lycopersicon esculentum plant which is homozygous for a single, recessive genetic factor which confers on the plant the ability to bear a mature fruit having a sucrose content from about 20 μmol per gram fresh weight to about 175 μmol per gram fresh weight, wherein the genetic factor is located adjacent a TG102 marker on chromosome
 3. 11. A plant according to claim 10 wherein the mature fruit has a sucrose content from about 50 μmol per gram fresh weight to about 150 μmol per gram fresh weight.
 12. A plant according to claim 10 wherein the mature fruit has a sucrose content of about 100 μmol per gram fresh weight.
 13. A plant according to claim 10 wherein the mature fruit has a total soluble solids content from about 6% by fresh weight to about 12% by fresh weight.
 14. A plant according to claim 10 wherein the mature fruit has a total soluble solids content from about 7.5% by fresh weight to about 10% by fresh weight.
 15. A plant according to claim 10 wherein the mature fruit has a total soluble solids content of about 8% by fresh weight.
 16. A plant according to claim 10 wherein the mature fruit has a level of invertase activity which is less than about 5 μmol of glucose produced per gram of fresh weight per minute.
 17. A plant according to claim 10 wherein the mature fruit has a level of invertase activity which is less than about 1 μmol of glucose produced per gram of fresh weight per minute.
 18. A plant according to claim 10 wherein the mature fruit has a level of invertase activity which is about 35 nmol of glucose produced per gram of fresh weight per minute.
 19. A plant according to claim 10 wherein the genetic factor is derived from a species in the genus Lycopersicon.
 20. A plant according to claim 10 wherein the genetic factor is derived from Lycopersicon chmielewskii.
 21. A Lycopersicon esculentum seed which is homozygous for a single, recessive genetic factor which confers on a plant derived from the seed the ability to bear a mature fruit having a sucrose content from about 20 μmol per gram fresh weight to about 175 μmol per gram fresh weight, wherein the genetic factor is located adjacent a TG102 marker on chromosome
 3. 22. A seed according to claim 21 wherein the mature fruit has a total soluble solids content from about 6% to about 12% by weight.
 23. A seed according to claim 21 wherein the mature fruit has a level of invertase activity which is less than about 5 μmol of sucrose produced per gram of fresh weight per minute.
 24. A seed according to claim 21 wherein the genetic factor is derived from Lycopersicon chmielewskii. 